1. Technical Field
The present invention relates to an assay and method for diagnosing disease. More specifically, the present invention relates to an immunoassay for use in diagnosing cancer.
2. Background Art
It is commonly known in the art that genetic mutations can be used for detecting cancer. For example, the tumorigenic process leading to colorectal carcinoma formation involves multiple genetic alterations (Fearon, et al. (1990) Cell 61, 759-767). Tumor suppressor genes such as p53, DCC and APC are frequently inactivated in colorectal carcinomas, typically by a combination of genetic deletion of one allele and point mutation of the second allele (Baker, et al. (1989) Science 244, 217-221; Fearon, et al. (1990) Science 247, 49-56; Nishisho, et al. (1991) Science 253, 665-669; and Groden, et al. (1991) Cell 66, 589-600). Mutation of two mismatch repair genes that regulate genetic stability was associated with a form of familial colon cancer (Fishel, et al. (1993) Cell 75, 1027-1038; Leach, et al. (1993) Cell 75, 1215-1225; Papadopoulos, et al. (1994) Science 263, 1625-1629; and Bronner, et al. (1994) Nature 368, 258-261). Proto-oncogenes such as myc and ras are altered in colorectal carcinomas, with c-myc RNA being overexpressed in as many as 65% of carcinomas (Erisman, et al. (1985) Mol. Cell. Biol. 5, 1969-1976), and ras activation by point mutation occurring in as many as 50% of carcinomas (Bos, et al. (1987) Nature 327, 293-297; and Forrester, et al. (1987) Nature 327, 298-303). Other proto-oncogenes, such as myb and neu are activated with a much lower frequency (Alitalo, et al. (1984) Proc. Natl. Acad. Sci. USA 81, 4534-4538; and D'Emilia, et al. (1989) Oncogene 4, 1233-1239). No common series of genetic alterations is found in all colorectal tumors, suggesting that a variety of such combinations can be able to generate these tumors.
Increased tyrosine phosphorylation is a common element in signaling pathways that control cell proliferation. The deregulation of protein tyrosine kinases (PTKS) through overexpression or mutation has been recognized as an important step in cell transformation and tumorigenesis, and many oncogenes encode PTKs (Hunter (1989) in oncogenes and the Molecular Origins of Cancer, ed. Weinberg (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), pp. 147-173). Numerous studies have addressed the involvement of PTKs in human tumorigenesis. Activated PTKs associated with colorectal carcinoma include c-neu (amplification), trk (rearrangement), and c-src and c-yes (mechanism unknown) (D'Emilia, et al. (1989), ibid; Martin-Zanca, et al. (1986) Nature 3, 743-748; Bolen, et al. (1987) Proc. Natl. Acad. Sci. USA 84, 2251-2255; Cartwright, et al. (1989) J. Clin. Invest. 83, 2025-2033; Cartwright, et al. (1990) Proc. Natl. Acad. Sci. USA 87, 558-562; Talamonti, et al. (1993) J. Clin. Invest. 91, 53-60; and Park, et al. (1993) Oncogene 8, 2627-2635).
Mutations, such as those disclosed above can be useful in detecting cancer. However, there have been few advancements which can repeatably be used in diagnosing cancer prior to the existence of a tumor. Approximately 40,500 new cases of head and neck squamous cell carcinoma (HNSCC) will be diagnosed in the United States and 11,170 Americans will die from this disease in the year 2006. Worldwide, HNSCC is the sixth most common malignancy with incidence of 644,000 new cases a year. Despite progress in diagnostic and treatment modalities in the past 30 years, long-term survival for patients affected by HNSCC has not significantly improved. One major impediment to improving survival in this patient population is the failure to detect this cancer at an early stage. More than two-thirds of patients with HNSCC are diagnosed at an advanced stage when the five-year survival is less than 40%. In many cases, these patients are offered radical treatments that often result in significant physical disfigurement as well as dysfunction of speech, breathing, and swallowing. The plight of these patients with advanced stage disease is in distinct contrast to that of patients who are diagnosed early. Early stage HNSCC patients have an excellent five-year survival rate of more than 80% and experience significantly less impact on their quality of life after treatment with single modality therapy. This dramatic difference in survival and quality of life underlies the importance of early detection in this disease.
Early detection can be achieved by screening patients at high risk for development of cancer. Although the American Cancer Society has issued guidelines for screening of breast, colon, prostate, and uterine cancers, no such guideline exists for HNSCC. This is especially unfortunate given that patients at increased risk for development of HNSCC can be easily identified (history of excess alcohol and/or tobacco use) and targeted for screening. Early detection can also be improved by reducing diagnostic delays, reported to be between 3 to 5.6 months. Misdiagnosis at initial presentation to primary care physicians is common (44% to 63%) and may be due to the nonspecific nature of presenting symptoms (sore throat, hoarseness, ear pain, etc) as well as the technical difficulty of examination in the head and neck region. The delay in diagnosis and referral to specialist has a significant negative impact on patient outcome and survival. Thus, there exists a need for a simple, noninvasive, and inexpensive test, widely accessible to physicians in the primary care setting, which can be used to screen (in asymptomatic patients) and diagnose (in symptomatic patients) HNSCC in high risk population to improve early detection.
Methods for detecting and measuring cancer markers have been recently revolutionized by the development of immunological assays, particularly by assays that utilize monoclonal antibody technology. Previously, many cancer markers could only be detected or measured using conventional biochemical assay methods, which generally require large test samples and are therefore unsuitable in most clinical applications. In contrast, modern immunoassay techniques can detect and measure cancer markers in relatively much smaller samples, particularly when monoclonal antibodies that specifically recognize a targeted marker protein are used. Accordingly, it is now routine to assay for the presence or absence, level, or activity of selected cancer markers by immunohistochemically staining tissue specimens obtained via conventional biopsy methods. Because of the highly sensitive nature of immunohistochemical staining, these methods have also been successfully employed to detect and measure cancer markers in smaller, needle biopsy specimens which require less invasive sample gathering procedures compared to conventional biopsy specimens. In addition, other immunological methods have been developed and are now well known in the art that allow for detection and measurement of cancer markers in non-cellular samples such as serum and other biological fluids from patients. The use of these alternative sample sources substantially reduces the morbidity and costs of assays compared to procedures employing conventional biopsy samples, which allows for application of cancer marker assays in early screening and low risk monitoring programs where invasive biopsy procedures are not indicated.
For the purpose of cancer evaluation, the use of conventional or needle biopsy samples for cancer marker assays is often undesirable, because a primary goal of such assays is to detect the cancer before it progresses to a palpable or detectable tumor stage. Prior to this stage, biopsies are generally contraindicated, making early screening and low risk monitoring procedures employing such samples untenable. Therefore, there is a general need in the art to obtain samples for cancer marker assays by less invasive means than biopsy, for example, by serum withdrawal.
Efforts to utilize serum samples for cancer marker assays have met with limited success, largely because the targeted markers are either not detectable in serum, or because telltale changes in the levels or activity of the markers cannot be monitored in serum. In addition, the presence of cancer markers in serum probably occurs at the time of micro-metastasis, making serum assays less useful for detecting pre-metastatic disease. Serological analysis of recombinant cDNA expression libraries (SEREX) of tumors with autologous serum is a well established technique which has been used successfully to identify many relevant tumor antigens. However, many of the SEREX antigens identified from a specific cancer patient reacted only with autologous serum antibodies from that particular patient and tend to recognize antibodies only at a low frequency in sera from other cancer patients. Thus, clinical tests based on these SEREX antigens that are patient-specific rather than cancer-specific are insufficient for early detection of cancer.
In view of the above, an important need exists in the art for more widely applicable, non-invasive methods and materials to obtain biological samples for use in evaluating, diagnosing and managing breast and other diseases including cancer, particularly for screening early stage, nonpalpable tumors. A related need exists for methods and materials that utilize such readily obtained biological samples to evaluate, diagnose and manage disease, particularly by detecting or measuring selected cancer markers, or panels of cancer markers, to provide highly specific, cancer prognostic and/or treatment-related information, and to diagnose and manage pre-cancerous conditions, cancer susceptibility, bacterial and other infections, and other diseases.
Autoantibodies against cancer-specific antigens have been identified in cancers of the colon, breast, kidney, lung, ovarian, and head and neck. Immune response with antibody production may be elicited due to the over-expression of cellular proteins such as Her2, the expression of mutated forms of cellular protein such as mutated p53, or the aberrant expression of tissue-restricted gene products such as cancer-testis antigens by cancer cells. Because these autoantibodies are raised against specific antigens from the cancer cells, the detection of these antibodies in a patient's serum can be exploited as diagnostic biomarkers of cancer in that particular patient. Further, the immune system is especially well adapted for the early detection of cancer since it can respond to even low levels of an antigen by mounting a very specific and sensitive antibody response. Thus, the use of immune response as a biosensor for early detection of cancer through serum-based assay holds great potential as an ideal screening and diagnostic tool.
With specific regard to such assays, specific antibodies can only be measured by detecting binding to their antigen or a mimic thereof. Although certain classes of immunoglobulins containing the antibodies of interest can, in some cases, be separated from the sample prior to the assay (Decker, et al., EP 0,168,689 A2), in all assays, at least some portion of the sample immunoglobulins are contacted with antigen. For example, in assays for specific IgM, a portion of the total IgM can be adsorbed to a surface and the sample removed prior to detection of the specific IgM by contacting with antigen. Binding is then measured by detection of the bound antibody, detection of the bound antigen or detection of the free antigen.
For detection of bound antibody, a labeled anti-human immunoglobulin or labeled antigen is normally allowed to bind antibodies that have been specifically adsorbed from the sample onto a surface coated with the antigen, Bolz, et al., U.S. Pat. No. 4,020,151. Excess reagent is washed away and the label that remains bound to the surface is detected. This is the procedure in the most frequently used assays, or example, for hepatitis and human immunodeficiency virus and for numerous immunohistochemical tests, Nakamura, et al., Arch Pathol Lab Med 1122:869-877 (1988). Although this method is relatively sensitive, it is subject to interference from non-specific binding to the surface by non-specific immunoglobulins that can not be differentiated from the specific immunoglobulins.
Another method of detecting bound antibodies involves combining the sample and a competing labeled antibody, with a support-bound antigen, Schuurs, et al., U.S. Pat. No. 3,654,090. This method has its limitations because antibodies in sera bind numerous epitopes, making competition inefficient.
For detection of bound antigen, the antigen can be used in excess of the maximum amount of antibody that is present in the sample or in an amount that is less than the amount of antibody. For example, radioimmunoprecipitation (“RIP”) assays for GAD autoantibodies have been developed and are currently in use, Atkinson, et al., Lancet 335:1357-1360 (1990). However, attempts to convert this assay to an enzyme linked immunosorbent assay (“ELISA”) format have not been successful. The RIP assay is based on precipitation of immunoglobulins in human sera, and led to the development of a radioimmunoassay (“RIA”) for GAD autoantibodies. In both the RIP and the RIA, the antigen is added in excess and the bound antigen:antibody complex is precipitated with protein A-Sepharose. The complex is then washed or further separated by electrophoresis and the antigen in the complex is detected.
Other precipitating agents can be used such as rheumatoid factor or C1q, Masson, et al., U.S. Pat. No. 4,062,935; polyethylene glycol, Soeldner, et al., U.S. Pat. No. 4,855,242; and protein A, Ito, et al., EP 0,410,893 A2. The precipitated antigen can be measured to indicate the amount of antibody in the sample; the amount of antigen remaining in solution can be measured; or both the precipitated antigen and the soluble antigen can be measured to correct for any labeled antigen that is non-specifically precipitated. These methods, while quite sensitive, are all difficult to carry out because of the need for rigorous separation of the free antigen from the bound complex, which requires at a minimum filtration or centrifugation and multiple washing of the precipitate.
Alternatively, detection of the bound antigen can be employed when the amount of antigen is less than the maximum amount of antibody. Normally, that is carried out using particles such as latex particles or erythrocytes that are coated with the antigen, Cambiaso, et al., U.S. Pat. No. 4,184,849 and Uchida, et al., EP 0,070,527 A1. Antibodies can specifically agglutinate these particles and can then be detected by light scattering or other methods. It is necessary in these assays to use a precise amount of antigen as too little antigen provides an assay response that is biphasic and high antibody titers can be read as negative, while too much antigen adversely affects the sensitivity. It is therefore necessary to carry out sequential dilutions of the sample to assure that positive samples are not missed. Further, these assays tend to detect only antibodies with relatively high affinities and the sensitivity of the method is compromised by the tendency for all of the binding sites of each antibody to bind to the antigen on the particle to which it first binds, leaving no sites for binding to the other particle.
For assays in which the free antigen is detected, the antigen can also be added in excess or in a limited amount although only the former has been reported. Assays of this type have been described where an excess of antigen is added to the sample, the immunoglobulins are precipitated, and the antigen remaining in the solution is measured, Masson, et al., supra and Soeldner, et al., supra. These assays are relatively insensitive because only a small percentage change in the amount of free antigen occurs with low amounts of antibody, and this small percentage is difficult to measure accurately.
Practical assays in which the free antigen is detected and the antigen is not present in excess of the maximum amount of antibody expected in a sample have not been described. However, in van Erp, et al., Journal of Immunoassay 12(3):425-443 (1991), a fixed concentration of monoclonal antibody was incubated with a concentration dilution series of antigen, and free antigen was then measured using a gold sol particle agglutination immunoassay to determine antibody affinity constants.
There has been much research in the area of evaluating useful markers for determining the risk factor for patients developing IDDM. These include insulin autoantibodies, Soeldner, et al., supra and circulating autoantibodies to glutamic acid decarboxylase (“GAD”), Atkinson, et al., PCT/US89/05570 and Tobin, et al., PCT/US91/06872. In addition, Rabin, et al., U.S. Pat. No. 5,200,318 describes numerous assay formats for the detection of GAD and pancreatic islet cell antigen autoantibodies. GAD autoantibodies are of particular diagnostic importance because they occur in preclinical stages of the disease, which can make therapeutic intervention possible. However, the use of GAD autoantibodies as a diagnostic marker has been impeded by the lack of a convenient, nonisotopic assay.
One assay method involves incubating a support-bound antigen with the sample, then adding a labeled anti-human immunoglobulin. This is the basis for numerous commercially available assay kits for antibodies such as the Syn ELISA kit which assays for autoantibodies to GAD65, and is described in product literature entitled “SynELISA GAD II-Antibodies” (Elias USA, Inc.). Substantial dilution of the sample is required because the method is subject to high background signals from adsorption of non-specific human immunoglobulins to the support.
Many of the assays described above involve detection of antibody that becomes bound to an immobilized antigen. This can have an adverse affect on the sensitivity of the assay due to difficulty in distinguishing between specific immunoglobulins and other immunoglobulins in the sample, which bind non-specifically to the immobilized antigen. There is not only a need to develop an assay that avoids non-specific detection of immunoglobulins, but there is also the need for an improved method of detecting antibodies that combines the sensitivity advantage of immunoprecipitation assays with a simplified protocol. Finally, assays that can help evaluate the risk of developing diseases are medically and economically very important. The present invention addresses these needs.